A primer-probe composition and its application

Abstract

The present invention relates to the technical field of gene detection. The present invention discloses a primer-probe composition and its application. The primer-probe composition of the present invention includes a primer set and a probe set. The primer set includes a first primer pair F1, R1, R2; a second primer pair F2, R3; and a third primer pair IC-F, IC-R. The probe set includes a first probe P1, P2, a second probe P3, and a third probe IC-P. The primer-probe composition of the present application can accurately detect the copy numbers of SMN1 and SMN2, with higher detection accuracy than that of conventional commercial kits, and good detection linearity. In combination with digital PCR technology, the LOB value is 2 copies / reaction, and the LOD value is 20 copies / μL reaction solution. It can be applied to the preparation of a high-accuracy SMA detection kit or preparation.

Description

Technical Field

[0001] The present invention relates to the technical field of gene detection, and particularly relates to a primer-probe composition and its application. Background Art

[0002] The statements in this section only provide background information related to the disclosure of the present application and may not constitute prior art.

[0003] Spinal muscular atrophy (SMA) is a group of hereditary neuromuscular diseases mainly characterized by muscle weakness and atrophy caused by the degeneration of α-motoneurons in the anterior horn of the spinal cord, and is the most common neuromuscular disease in children. SMA is an autosomal recessive genetic disease, with a carrier rate of 1 / 50 - 1 / 40 and an incidence rate of 1 / 10000 - 1 / 6000 live births. If both parents are carriers, the probability of a newborn being affected is 1 / 4.

[0004] SMA is related to the SMN (Survival of motor neurons) gene. There are two highly homologous copies of SMN, namely SMN1 and SMN2, with only 5 base differences between them. Two of these bases are located in exons 7 and 8, and the other three bases are in introns 6 and 7. Homozygous deletion of exon 7 of the SMN1 gene is the main pathogenic cause of SMA. In healthy individuals, this gene produces the survival motor neuron protein or SMN protein, which is crucial for controlling the nerve function of muscles. If exon 7 of SMN1 is deleted, SMN protein cannot be synthesized, resulting in the inability of nerve cells to function properly and ultimately die, leading to muscle weakness and affecting a person's daily life and even life. An increase in the copy number of the SMN2 gene is beneficial to the alleviation of SMA symptoms. The copy number of SMN2 in each person may be 0, 1, 2, 3, or 4. SMN2 is currently recognized as an SMA modifier. The more copies of SMN2 a patient carries, the milder the phenotype. Although the correlation with the phenotype is not completely consistent, the copy number of SMN2 is still regarded as one of the standard steps for SMA diagnosis in domestic and international management consensus.

[0005] Detection of the copy numbers of SMN1 and SMN2 can be used for gene diagnosis of diseases, carrier screening, prenatal diagnosis of infants, preimplantation genetic testing, etc. It can help confirm the disease type of patients during the disease diagnosis process for precise medication, help identify individuals without symptoms but carrying pathogenic gene mutations, thereby providing genetic counseling and reproductive choices for families, screening healthy embryos in assisted reproduction, and reducing the risk of genetic diseases being passed on to the next generation. It not only contributes to the early diagnosis and prevention of diseases, but also provides important genetic reference information for individuals and families.

[0006] The existing technologies for detecting spinal muscular atrophy include: first, chromosome karyotype analysis, to determine whether there are abnormal chromosomes; mitochondrial DNA detection: to determine whether there are abnormal mitochondria. These two methods are relatively subjective in judging the results and are prone to missed detection and wrong detection. Secondly, there is conventional gene mutation detection, which determines whether it is caused by gene mutation by detecting gene sequence. This method is relatively cumbersome to operate. There is also a serum detection method, which detects the content of specific enzymes in serum, but this method is an indirect detection and can only assist in determining whether there is spinal muscular atrophy; the quantification of SMN gene copy number has become the current mainstream method, including DHPLC and other technologies. The principle of this method is that after DNA is denatured and gradually cooled and annealed, the heterozygous and wild-type PCR amplification products form homologous duplexes while also mismatching to form heteroduplexes. Under partial denaturation conditions, mismatched heteroduplex DNA is more likely to melt into single-stranded DNA, and the binding force with the DNA elution column is reduced. It is easier to be eluted by the eluent than homologous duplex DNA molecules, thereby separating from homologous duplex DNA. However, the disadvantage is that this method is limited by primers, reaction conditions, temperature when binding to the column and other factors, resulting in poor stability of the results.

[0007] There are currently a number of test kits on the market that use qPCR and related methods to measure exon 7 of SMN1 and SMN2, with a detection limit as low as 0.25 ng / μL. However, using qPCR and related methods for copy number detection has the problem of low accuracy of the results. Summary of the invention

[0008] The purpose of the present invention is to provide a primer probe combination and a kit using the same in view of the deficiencies of the current prior art. The probe and primer combination of the present invention are designed for special sites of SMN1 and SMN2. The site is located at the differential base (c.840C>T) in the coding region of exon 7 and the differential base (c.239G / A) in the coding region of exon 8 for detection, and the special design of the primer probe makes the binding ability of the primer probe stronger, and the selection of the primer and probe sequence fragments makes the primer and probe less likely to form a loop structure, so as to achieve the purpose of absolute quantification of SMN1 and SMN2 genes, and the quantitative results are stable and reliable.

[0009] The technical solution of the present invention is as follows:

[0010] A primer-probe combination, comprising a primer set and a probe set,

[0011] The primer set includes a first primer pair, a second primer pair complementary to the SMA pathogenic gene region, and a third primer pair complementary to the housekeeping gene in cells; the sequences of the first primer pair are F1, R1, and R2 shown in SEQ ID NO.1-3, the sequences of the second primer pair are F2 and R3 shown in SEQ ID NO.6 and SEQ ID NO.7, and the sequences of the third primer pair are IC-F and IC-R shown in SEQ ID NO.9 and SEQ ID NO.10;

[0012] The probe set includes a first probe, a second probe complementary to the SMA pathogenic gene region, and a third probe complementary to the housekeeping gene in cells; the first probes are P1 and P2 with sequences shown in SEQ ID NO.4 and SEQ ID NO.5, the sequence of the second probe is P3 shown in SEQ ID NO.8, and the sequence of the third probe is IC-P shown in SEQ ID NO.11.

[0013] Preferably, the first probe, the second probe, and the third probe are respectively labeled with different fluorescent markers. The fluorescent markers are selected from fluorescent dyes with maximum emission wavelengths of 518nm, 538 - 555nm, 574 - 575nm, 602 - 615nm, 640nm, 660 - 667nm, or 690nm.

[0014] Preferably, the first primer pair and the first probe specifically amplify the differential base (c.840C>T) region of exon 7 of SMN1; the second primer pair and the second probe specifically amplify the differential gene (c.239G / A) region of exon 8 of SMN1. The 5' end part (the base to the left of the middle modified base) of the first primer pair design and the first probe contains 16 bases, which have no complementary sequence with the amplified product target sequence, but are complementary to the sequence at the 5' end of the corresponding upstream primer. The 3' end part (the base to the right of the middle modified base) of the probe contains 16 bases, which are complementary to the target sequence of the amplified product. The 5' end of the upstream primer contains a sequence that does not pair with the target sequence of the amplified product but is complementary to the 5' end part of the probe. To improve the specificity of the probe, the region of the 3' end of the probe complementary to the template strand is designed to be relatively short. When the upstream and downstream primers bind to the template and amplify to form a product, the probe specifically binds to the fully complementary template product with the help of the binding of the 5' end to the complementary strand of the upstream primer, while the product with a mismatch does not bind, to distinguish the mutant types in the (c.840C>T) region and the (c.239G / A) region.

[0015] Another aspect of the present invention provides the application of the primer-probe composition as described above in the preparation of a spinal muscular atrophy (SMA) gene mutation detection preparation and / or kit.

[0016] A kit for detecting spinal muscular atrophy (SMA) gene mutations, comprising the primer-probe composition as described above.

[0017] According to a preferred embodiment, it further comprises a universal digital PCR reaction premix (DNA, 2×), an enhancer, a blank control, a negative control, and a positive control.

[0018] On the other hand, the present invention provides a method for detecting spinal muscular atrophy (SMA) gene mutations using a primer-probe composition as described above in the preparation of a kit for detecting spinal muscular atrophy (SMA) gene mutations:

[0019] Step (1): Obtain a biological sample from the subject to be tested;

[0020] Step (2): Use the probe and primer composition to perform fluorescence quantitative PCR on the biological sample to determine whether there is a spinal muscular atrophy (SMA) mutant gene.

[0021] According to a preferred embodiment, the biological sample is selected from venous blood.

[0022] Compared with the existing technology, the beneficial effects of the present invention are:

[0023] 1. A primer-probe composition and its application can perform absolute quantification of the gene copy number, and obtain the relative copy numbers of SMN1 and SMN2 through the ratio with the internal reference gene, maximizing the avoidance of missed detection due to integration loss; and through the design of primers and probes at specific sites and the addition of an enhancer, SMN1 and SMN2 can be effectively distinguished, solving the problem of non-specific amplification caused by the high homology of the two genes.

[0024] 2. A primer-probe composition and its application: ① High sensitivity: It can detect extremely trace nucleic acid samples in a complex background; ② Strong specificity: It can effectively distinguish between SMN1 and SMN2 genes, and the detection of SMN1 is not interfered by the high background of SMN2; ③ Absolute quantification: It can perform absolute quantification of the test sample without a standard; ④ Low sample requirement, and can detect various specimen types; ⑤ High tolerance: It reduces the influence between reaction systems and the interference of background sequences and inhibitors on the reaction. These changes improve the detection sensitivity of this project and reduce the probability of missed detection and false positives. ⑥ Dual-gene detection: In addition to detecting the SMN1 gene, digital PCR can also detect the copy number of the SMN2 gene, providing more information for disease diagnosis and treatment. Description of the Drawings

[0025] Figure 1 It is the linear regression equation of Example 2 of this application;

[0026] Figure 2SD, CV, and adv. results of Example 2 of this application;

[0027] Figure 3 Statistical graph of data of the NC group and the PC group for the determination of LOD performance in Example 3 of this application;

[0028] Figure 4 Statistical graph of data of the NC group and the PC group for the determination of precision in Example 4 of this application;

[0029] Figure 5 Detection data result graph of the thermal stability test in the reagent stability test of Example 5 of this application;

[0030] Figure 6 Detection data result graph of the freeze - thaw stability test in the reagent stability test of Example 5 of this application. Detailed implementation manners

[0031] The specific examples listed in the present invention are only examples of the present invention, and the present invention is not limited to the specific examples described below. For those skilled in the art, any equivalent modifications and substitutions to the following described examples are also within the scope of the present invention. Therefore, equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention. For those conditions not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. All reagents or instruments not specified by the manufacturer are conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are given in the following detailed implementation manners. Those skilled in the art should understand that the present invention can still be implemented without some specific details. In other examples, methods, means, equipment, and steps well - known to those skilled in the art are not described in detail to highlight the gist of the present invention.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Without special instructions, the units used in this specification are all international standard units, and the numerical values and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0033] In the phenotypically normal population (including normal individuals and carriers), the copy number of the SMN1 gene is usually 1 to 4. The genotypes of normal individuals include the common [1+1] type and the rare [2+1] type and [2+2] type; in carriers, one chromosome contains 1 or 2 copies of the functionally normal SMN1 gene, and the other chromosome contains a functionally abnormal SMN1 gene with a deletion or a minor variation. The genotypes can be heterozygous deletions ([1+0] type, [2+0] type) or heterozygous mutations ([1+1d] and [2+1d] type) [d represents a minor variation within the SMN1 gene resulting in abnormal function, such as nonsense mutations, frameshift mutations, missense mutations, etc.]. In SMA patients with abnormal phenotypes, there are mainly two types of mutant genotypes. 95% are caused by homozygous deletion of both alleles of SMN1 ([0+0] genotype); 5% are caused by compound heterozygous mutations of SMN1 ([0+1d] genotype), and the situation where both alleles of SMN1 are minor variations ([1d+1d]) is very rare. Most of the SMN1 deletions are the common deletion of exon 7 and exon 8, and a small part is only the deletion of exon 7.

[0034] The copy number of SMN2 ranges from 0 to multiple. The copy number of SMN2 is currently recognized as a modifier of SMA. The more copies of SMN2 a patient carries, the milder the phenotype, but its correlation with the phenotype is not completely consistent. Sequence variations of SMN2 and other genes may also affect the phenotype of SMA. Therefore, the copy number result of SMN2 can only provide a possible reference information for the clinical severity of SMA-affected children or fetuses rather than a definite conclusion. In addition, the copy number detection of SMN2 is not required for non-patient populations. At the same time, it should be noted that both SMN1 and SMN2 are located at 5q13.2, and there are multiple pairs of homologous genes including SMN in this region, which leads to unequal genomic exchange and gene conversion, resulting in various changes in the copy numbers of SMN1 and SMN2.

[0035] Since the pathogenic genes of SMA, SMN1 and SMN2, are DNA, the invention uses the polymerase chain reaction method with end-point fluorescence monitoring, and uses the designed primer-probe composition to detect the differential bases (c.840C>T) in the coding region of exon 7 and the differential bases (c.239G / A) in the coding region of exon 8, perform absolute quantification of SMN1 and SMN2, and obtain the relative copy numbers of SMN1 and SMN2 through the ratio with the reference gene.

[0036] The features and performance of the present invention will be further described in detail below in conjunction with the embodiments.

[0037] Example 1

[0038] Use the primer-probe composition of the present invention for SMA detection to determine the detection accuracy of the primer-probes of the present application.

[0039] The primer-probe composition sequence included in the present invention is as follows in the sequence listing:

[0040]

[0041] The components of the kit included in the present invention are as follows in the reagent composition table:

[0042]

[0043] The following are the performance detection indexes of the PCR amplification reagent for the present invention

[0044] Limit of blank: Detecting clinical negative samples, the detection result is negative.

[0045] Limit of detection (LOD): The lowest detection concentration that the detection method can detect. In this test, a positive detection rate of ≥95% (19 / 20) is used as the standard for determining the LOD.

[0046] Precision: Detecting the precision reference product, the within-batch precision meets the coefficient of variation (CV, %) of the concentration value ≤10.0%.

[0047] Linearity: Sample gradient dilution amplification, linear regression R 2 ≥0.98 is used as the qualified standard for evaluating the reagent.

[0048] Stability: Thermal acceleration stability, freeze-thaw stability.

[0049]

[0050] Determination scheme for LOB performance

[0051] (1) Detect 20 negative samples (blank control products) with 1 batch of reagent;

[0052] (2) Standard: Sort the experimental results from small to large, and take the value of 95% as the LOB value;

[0053] (3) Instrument and equipment: Mike Bio D600 digital PCR analysis system.

[0054] Detection result:

[0055]

[0056] Conclusion

[0057] (1)Perform 24 repeated detections on the blank control. Among them, there are 13 groups of false-positive droplets in the FAM channel (SMN1 exon7), 12 groups of false-positive droplets in the HEX channel (RPPH1), 11 groups of false-positive droplets in the ROX channel (SMN2 exon7), and 9 groups of false-positive droplets in the Cy5 channel (SMN1 exon8), with 1-2 false-positive droplets per reaction.

[0058] (2)Conclusion: According to the test result data, the LOB value is 2 copies / reaction, that is, the blank control has no more than 2 positive droplets per reaction.

[0059] Example 2

[0060] Linear range performance verification plan

[0061]

[0062] Specific steps:

[0063] (1)Select the template: Select the human genomic standard with normal genotype as the test sample.

[0064] (2)Determine the stock solution concentration: Take the high-concentration template to be measured, perform 10-fold gradient dilution, measure the concentrations of two different concentrations of the template, dilute the high-concentration template to 10000 copies / μL according to the measured value, and then start 2.5-fold gradient dilution for 8 gradients, as shown in the following table;

[0065]

[0066] (3)Determine the linear range: Add the diluted template to the reaction system at a volume of 2.5 ul per 25 ul reaction system for measurement, and measure 3 replicates for each concentration gradient;

[0067] (4)Analyze the measurement results: Take the average value of the measured results for linear analysis;

[0068] (5)Instrument and equipment: Mike Bio D600 digital PCR analysis system

[0069] Results

[0070]

[0071] Take the average value of the data of the repeated groups to obtain the following data:

[0072]

[0073] Perform linear calculation on the above data, and the obtained linear regression equation is as Figure 1 shown. Calculate SD, CV, and adv. for the detection data, and the results are asFigure 2 as shown

[0074] Conclusion

[0075] (1)According to the data, the linear Rs of the FAM, HEX, ROX, and Cy5 channels are 0.9988, 0.9982, 0.9986, and 0.9988 respectively, all of which are greater than or equal to 0.99, indicating that the primer-probe composition has good detection linearity in the concentration range of 1.64 copies / μL - 1000.00 copies / μL. 2 respectively, all of which are greater than or equal to 0.99, indicating that the primer-probe composition has good detection linearity in the concentration range of 1.64 copies / μL - 1000.00 copies / μL.

[0076] (2)According to the data (CV heat map), it can be preliminarily determined that the LOD of the system for detecting RCNs is 25.6 copies / μL of the reaction solution, that is, 625 ± 100 copies / reaction, providing a reference for the next LOD test.

[0077] Example 3 Determination of LOD Performance

[0078] Scheme

[0079]

[0080] (1)Template preparation for determination: Select the positive and negative quality control products diluted to 500 copies / μL in the linear test for LOD test respectively;

[0081] (2)LOD preliminary experiment: According to the linear results of Example 2, select samples of 300, 400, and 500 copies / reaction for 8 repeated experiments respectively. If the RCNs of the samples can be accurately measured in all 8 experiments and the quantitative CV values of each channel are within 10%, then this sample can be used for the formal LOD determination; if none of the 3 groups of concentrations meet the above requirements, the concentration can be increased by 100 copies for the formal LOD determination. Finally, through the preliminary experiment, it is determined that 500 copies / reaction meets the test standard and can be used for the formal LOD test.

[0082] (3)LOD determination: Measure according to the sample volume of 1.25 μL added to each 25 μL reaction system, and repeat each template 20 - 24 times. Finally, use a positive detection rate of ≥ 95% (23 / 24) as the standard for determining LOD; (the detection standard for the NC quality control product is that the RCNs of exons 7 and 8 of SMN1 (i.e., S1E7, S1E8) are both 2 copies, and SMN2 is also 2 copies; the positive detection standard for the PC quality control product is that the RCNs of exons 7 and 8 of SMN1 (i.e., S1E7, S1E8) are both 0 copies, and SMN2 is multi-copy (> 2 copies, theoretically 4 copies))

[0083] (4)Instrument and equipment: Mike Bio D600 Digital PCR Analysis System

[0084] Results

[0085]

[0086]

[0087] The statistical charts of the data of the NC group and the PC group are as Figure 3 shown

[0088] Results

[0089] (1)The SMN copy number variation detection reagent was used to perform 24 repeated detections on two groups of quality control products with a reaction solution concentration of 20 copies / μL, and the detection rates were 100% and 100% respectively, meeting the measurement standard of LOD

[0090] However, the relative copy number quantification of SMN2 in the PC quality control product deviated significantly from the theoretical value (on the low side). Considering that this quality control product is an artificial plasmid synthesis template with a large number of repetitive sequences in the design and difficult synthesis, it may lead to low template quality. In addition, the samples need to be digested and diluted, resulting in a large relative copy number deviation among the targets

[0091] (2)Conclusion: According to the test result data, LOD = 20 copies / μL reaction solution (i.e., 500 copies / reaction)

[0092] Determination of precision in Example 4

[0093] Scheme

[0094] (1)Template preparation for determination: Quantify two groups of templates with different genotypes (NC, PC). Generally, select templates with a concentration of 10 3 -10 4 copies / μL, and then dilute the template to 1500 copies / μL according to the actual quantification result as the template for precision determination

[0095] (2)Precision determination: Add 2 μL of the sample volume to the 1500 copies / μL template according to each 25 μL reaction system for determination, that is, 120 copies / μL reaction solution (3000 copies / reaction). Each group of templates is repeated 12 times. Finally, the coefficient of variation (CV, %) of the template quantitative detection result ≤ 10.0% is used as the standard for evaluating the precision of the kit

[0096] (3)Instrument and equipment: Mike Bio D600 Digital PCR Analysis System

[0097] Results:

[0098]

[0099]

[0100] The statistical charts of the data of the NC group and the PC group are as Figure 4 shown below.

[0101] Conclusion

[0102] (1)The CV values of the absolute quantification and RCNs of each group in the two groups of templates were less than 5% in the range of 2000 - 3000 copies / reaction, which were within the acceptable range;

[0103] (2)The precision performance of this reagent is good.

[0104] Example 5 Reagent Stability

[0105] Scheme

[0106]

[0107] Specific steps:

[0108] (1)Reagent preparation: The reagents that need to be accelerated include 2X mix, 10X primer-probe mix and Taq enzyme, a total of three kinds. Each experiment requires 12 times. Heat acceleration is carried out according to the packaging specifications. Since the minimum specification is 24 times, 3 batches of experimental reagents with a 24-time specification are prepared;

[0109] (2)Reagent aliquoting: The prepared reagents are aliquoted into 3 storage tubes provided by Mike, and the experimental amount for each reagent is 24 times, as follows:

[0110]

[0111] (3)Heat acceleration: According to the following table, the 3 aliquotted reagents are respectively put into small self-sealing bags and placed in a 37°C water bath for heating and storage according to the specific date, and other reagents are stored in a -20°C refrigerator;

[0112]

[0113] (4)Freeze-thaw experiment: According to the following table, the 3 aliquotted reagents are respectively put into small self-sealing bags and the freeze-thaw test is carried out according to the specific date, and other reagents are stored in a -20°C refrigerator. The method of freeze-thaw is to place at room temperature for a certain time to fully thaw, and then put it into a -20°C refrigerator overnight to fully freeze;

[0114]

[0115] (5)Verification by thermal acceleration experiment: Take 3 batches of reagents after the acceleration is completed, plus the control reagent, and conduct the determination according to the following method;

[0116] First, conduct experiments on the reagents placed at -20°C and those accelerated at 37°C for 3 days and 5 days. If there are no problems with the experimental data of the reagents accelerated at 37°C for 3 days and 5 days compared with the determination results of the reagents placed at -20°C, the experiment of accelerating at 37°C for 7 days can be carried out. If there are problems with the data of accelerating at 37°C for 5 days, the experimental verification of accelerating at 37°C for 7 days can be omitted;

[0117] (6)Verification by freeze-thaw experiment: Take 3 batches of reagents after the acceleration is completed, plus the control reagent, and conduct the determination according to the following method;

[0118] First, conduct experimental verification on freeze-thaw for 5 days, 7 days and the -20°C control system. If there is no difference between the results of freeze-thaw for 5 days and 7 days and the results of the -20°C control system, the experiment of freeze-thaw for 3 days can be omitted; if there are problems with the results of freeze-thaw for 5 days and 7 days, the experiment of freeze-thaw for 3 days can be carried out;

[0119] Results

[0120] (1)Thermal stability test (copies / μL reaction solution, 25 μL)

[0121]

[0122] The detection results are as Figure 5 shown.

[0123] (2)Freeze-thaw stability test (copies / μL reaction solution, 25 μL)

[0124]

[0125] The detection results are as Figure 6 shown ( Figure 6 The horizontal and vertical coordinates of Figure 5 are in the same order as those in

[0126] Conclusions

[0127] (1)Thermal stability

[0128] For the reagent incubated at 37°C for 3 / 5 / 7 days, the lowest detection limit, the NC and PC detection rates are all 100%;

[0129] For the reagent incubated at 37°C for 3 / 5 / 7 days, the quantitative values of each target gene are consistent with those of the control group.

[0130] This reagent has good thermal stability and can be transported at room temperature for a short time.

[0131] (2)Freeze-thaw stability

[0132] The lowest detection limits of the reagent after freeze-thawing 3 / 5 / 7 times showed 100% detection rates for both NC and PC.

[0133] The quantification of the reference gene and target gene of the reagent within 7 times of freeze-thawing was consistent with that of the control group.

[0134] The reagent has good freeze-thaw stability.

[0135]

[0136] Example 6 A Probe and Primer Composition

[0137] A probe and primer composition, which comprises the following components:

[0138] (1) Probe set, including a first probe complementary to the mutant gene region of spinal muscular atrophy (SMA), P1 and P2 shown in SEQ ID NO.4 and SEQ ID NO.5; a second probe complementary to the mutant gene region of spinal muscular atrophy (SMA), P3 shown in SEQ ID NO.8; a third probe complementary to the DNA of the intracellular housekeeping gene, IC-P shown in SEQ ID NO.11.

[0139] (2) Primer set, including a first primer pair complementary to the mutant gene region of spinal muscular atrophy (SMA), F1, R1, R2 shown in SEQ ID NO.1 - 3; a second primer pair complementary to the mutant gene region of spinal muscular atrophy (SMA), F2, R3 shown in SEQ ID NO.6 and SEQ ID NO.7; a third primer pair complementary to the housekeeping gene, IC-F, IC-R shown in SEQ ID NO.9 and SEQ ID NO.10.

[0140] The first probe is labeled with a fluorescent dye with a maximum emission wavelength of 522 nm, the second probe is labeled with a fluorescent dye with a maximum emission wavelength of 602 nm, and the third probe is labeled with a fluorescent dye with a maximum emission wavelength of 664 nm.

[0141] Using the first probe and the first primer pair to amplify the template of the mutant gene region of spinal muscular atrophy (SMA) on ABI7500; using the second probe and the second primer pair to amplify the template of the mutant gene region of spinal muscular atrophy (SMA) on ABI7500; using the third probe and the third primer pair to amplify the template of the intracellular housekeeping gene on ABI7500. Both the probe set and primer set of the present invention can effectively amplify the relevant nucleotide sequences.

[0142] Example 7

[0143] A kit for detecting the mutation gene region of spinal muscular atrophy (SMA), which kit includes the probe set and primer set described in Example 6.

[0144] Using this kit, amplification is carried out on the mixture of the template of the mutation gene region of spinal muscular atrophy (SMA) and the template of the housekeeping gene in cells on ABI7500. It can be seen that this kit can detect gene mutations in the mutation gene region of spinal muscular atrophy (SMA), and at the same time detect the template of the housekeeping gene in cells. This housekeeping gene in cells is used to evaluate the sample quality and PCR inhibition factors.

[0145] The positive control of this kit is a mixed plasmid of the mutation gene region template and the housekeeping gene template in cells of patients clinically diagnosed with spinal muscular atrophy (SMA); the negative control is water.

[0146] Example 8

[0147] To detect the accuracy and effectiveness of the kit described in Example 1 of the present invention in diagnosing spinal muscular atrophy (SMA), two existing commercially available similar kits (hereinafter referred to as Kit A (Wuseshi) and Kit B (Tianlong) respectively) and the kit of the present invention (hereinafter simply referred to as "the present invention") are used to conduct a comparative test on 100 clinical samples at the same time to evaluate the consistency of the test system and the control system. 3.1

[0149] (1) Nucleic acid preparation

[0150] Nucleic acid extraction generally can be divided into four steps: lysis - binding - washing - elution

[0151] (2) Nucleic acid amplification (digital PCR method)

[0152] Nucleic acid amplification generally can be divided into: preparation of PCR amplification reagents (reagent preparation area) - loading of nucleic acid samples (sample preparation area) - droplet preparation and PCR amplification (amplification area)

[0153] (3) Data analysis

[0154] 3.2 Sample processing (performed in the sample processing area)

[0155]

[0156] (1)Sample nucleic acid extraction

[0157] The sample is a venous blood sample; among them, the negative control product should participate in the parallel extraction step of nucleic acid.

[0158] ① Pipette 10 μL of the pretreatment solution into a 1.5 mL EP tube, add 400 μL of the clinical sample, and mix well. Then add 400 μL of Lysis buffer and 8 μL of magnetic beads, mix well, let stand for 10 min, and centrifuge briefly.

[0159] ② Place the EP tube on the magnetic rack for 2 min, and aspirate the supernatant.

[0160] ③ Add 750 μL of Wash buffer I and 50 μL of Wash buffer II, mix well, and centrifuge briefly.

[0161] ④ Place the EP tube on the magnetic rack for 2 min, aspirate the supernatant, and centrifuge briefly.

[0162] ⑤ Place the EP tube on the magnetic rack for 2 min, aspirate the residual liquid, and air-dry for 2 min.

[0163] ⑥ Add 35 μL of Elution buffer, mix well, and centrifuge briefly.

[0164] ⑦ Place the EP tube in a 60 °C metal bath and incubate for 10 min.

[0165] ⑧ Place the EP tube on the magnetic rack for 2 min, aspirate the supernatant, and discard the EP tube containing the magnetic beads.

[0166] (2) Sample Mixing

[0167] Take N PCR reaction tubes, add 15 μL of the PCR reaction system prepared in step 3.2 to each tube, and then add 5 μL of the positive control product, the negative control product obtained by treating in step 3.2 (1), and the supernatant of the test sample respectively. Cover the tube caps, centrifuge briefly, and transfer to the amplification area.

[0168] 3.3 PCR Amplification (Performed in the amplification area)

[0169] (1) Place each reaction tube into the fluorescence PCR detector and set the sample name;

[0170] (2) Set the PCR reaction conditions according to the following table;

[0171]

[0172] (3) Set the reaction volume to 20 μL;

[0173] (4) Instrument detection channel selection: Select the FAM, VIC, ROX, and CY5 channels. The spinal muscular atrophy (SMA) gene mutations represented by each channel are as follows in the table:

[0174]

[0175] (5)After the settings are completed, save the file and run the reaction program; after the PCR program is completed, save the results, and dispose of the reaction tubes and other waste according to the management rules of the PCR laboratory.

[0176] (6)Quality control: There should be no Ct values in all channels of the negative control product; otherwise, it indicates contamination. For the positive control product, the Ct values of both the FAM and ROX channels should be ≤ 32; otherwise, it indicates degradation of the positive control product or invalidation of the amplification reagent, as shown in the following table.

[0177]

[0178] 3.4 Interpretation of test results

[0179] (1)Quality control standards

[0180] ① Threshold: The automatic setting of the instrument is 10 times the standard deviation of the fluorescence signal in 3 to 15 cycles. The principle of manual setting is that the threshold line just exceeds the highest point of the curve of the negative control product (irregular noise line) and the fluorescence background value of the sample. Try to select the initial stage of the exponential phase.

[0181] ② Baseline: The starting point of the baseline should avoid selecting the first few cycles (signal increase due to high temperature), generally between 3 and 6 cycles. The end point should avoid selecting the area where the signal has started to increase significantly, generally select the point 3 to 4 cycles less than the smallest Ct value in this group. It is recommended to have an interval of more than 8 cycles between the starting point and the end point, and then analyze the results.

[0182] ③ Only when the amplification curve shows a typical S shape can it be considered amplification positive; otherwise, it is considered amplification negative. If the reaction result is in the effective area (the effective determination refers to the test method and the positive judgment value), the result can be further analyzed; otherwise, the test is invalid.

[0183] ④ Result reporting

[0184] If the Ct value of the FAM signal of the test sample is ≤ 32, it is reported as positive for spinal muscular atrophy (SMA) gene mutation;

[0185] If the Ct value of the ROX signal of the test sample is ≤ 32, it is reported as positive for spinal muscular atrophy (SMA) gene mutation;

[0186] If there are no Ct values for FAM, VIC, and ROX, it is reported as negative; if the Ct value of the test sample > 32, it is in the gray area and needs to be retested. If the Ct value of the retest is no value or > 32, it is reported as negative; if the Ct value of the retest ≤ 32, it is reported as positive.

[0187] ⑤ Test results

[0188] The specific results are shown in the following table:

[0189]

[0190] The detection results show that the detection results of the kit of the present invention for the gene mutation of spinal muscular atrophy (SMA) are relatively consistent with the positive coincidence rates of invention kits A and B. In terms of sensitivity, it is superior to similar inventions that are detected in two tubes and similar inventions without internal control quality control. The sensitivity, specificity, and accuracy of the kit of the present invention are all superior to those of kit A and kit B.

[0191] Example 9

[0192] The 1st clinical sample was detected as a negative sample for the gene mutation of spinal muscular atrophy (SMA) using kits A and B, while the kit of the present invention detected it as a positive sample for the gene mutation of spinal muscular atrophy (SMA); the 9th clinical sample was detected as a negative sample for the gene mutation of spinal muscular atrophy (SMA) using kits A and B, while the kit of the present invention detected it as a positive sample for the gene mutation of spinal muscular atrophy (SMA).

[0193] In order to further verify the positive results detected by the kit of the present invention, for the samples (the 1st and 9th clinical samples) whose comparison results between the present invention and kits A and B do not fully match, through amplification with gene primers and probes in the L1 region, the PCR products that obtained positive signals were verified by sequencing using specific primers by the sanger method, and the sequencing was handed over to a third-party sequencing company.

[0194] PCR product sequencing results: The sequences obtained by sequencing were compared by NCBI Blast, and it was confirmed that all the samples detected as positive by the present invention but detected as negative by the comparison reagents were true positives and the typing was correct. The Blast comparison results after sequencing the 1st clinical sample showed that the 1st sequence was a gene fragment of the gene mutation of spinal muscular atrophy (SMA); the Blast comparison results after sequencing the 9th clinical sample showed that the 9th sequence was a gene fragment of the gene mutation of spinal muscular atrophy (SMA).

[0195] Combined with the above experimental results, the coincidence rate of the detection results of the kit of the present invention with those of kits A and B is relatively consistent, and there is a certain advantage in sensitivity, indicating that the kit of the present invention can not only detect the gene mutation of spinal muscular atrophy (SMA), but also conduct quality control through the detection of housekeeping genes in cells, meet the screening requirements and can better provide clinical guidance, and can largely prevent missed detections caused by insufficient sensitivity or viral base mutations.

[0196] It should be understood that the disclosed invention is not limited solely to the specific methods, protocols, and materials described, as these may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims.

[0197] Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. These equivalents are also encompassed by the appended claims.

[0198] Advantages of primer and probe design:

[0199] ‌High specificity and secondary structure considerations‌: The probe design needs to be highly specific. Primers and probes are designed for specific regions of SMN1 and SMN2 to avoid non-specific amplification caused by their close homology. At the same time, amplification fragments with the minimum secondary structure are selected to reduce reaction hindrance. If secondary structure cannot be avoided, the annealing temperature needs to be increased accordingly; strictly control the ‌amplification length and GC content‌: The amplification length should be controlled within a certain range (such as 50 - 150 bp) to maintain the consistency of analysis; the GC content needs to be maintained within an appropriate range (such as 30% - 80%) to avoid non-specific reactions and signal interference, ‌avoid repetitive sequences‌: To ensure efficiency and repeatability, repetitive nucleotide sequences, especially consecutive Gs, should be avoided to prevent the formation of primer dimers and hairpin structures, so as to ensure the specificity and accuracy of the reaction and avoid missed and misdetected cases. The molecular beacon probe is also used for probe design. Its working principle is based on a stem-loop dual-labeled oligonucleotide probe with a hairpin structure. When paired with the template, the fluorophore and quencher are separated, generating a fluorescence signal, making the signal-to-noise ratio higher.

[0200] The above-described embodiments merely represent specific embodiments of the present application. Their descriptions are relatively specific and detailed, but they should not be construed as limiting the protection scope of the present application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the technical solution of the present application, several modifications and improvements can still be made, and these all fall within the protection scope of the present application.

Claims

1. A primer-probe composition, characterized in that, it comprises a primer set and a probe set, the primer set includes a first primer pair, a second primer pair complementary to the SMA pathogenic gene region, and a third primer pair complementary to the intracellular housekeeping gene; the probe set includes a first probe, a second probe complementary to the SMA pathogenic gene region, and a third probe complementary to the intracellular housekeeping gene; the first primer pair and the first probe specifically amplify the differential base c.840C>T region of exon 7 of SMN1; the second primer pair and the second probe specifically amplify the differential gene c.239G / A region of exon 8 of SMN1; the first primer pair includes F1, R1, R2, and the sequences are as shown in SEQ ID NO.1-3; the second primer pair includes F2, R3, and the sequences are as shown in SEQ ID NO.6 and SEQ ID NO.7; the first probe includes probe P1 and P2, and the sequences of the probe P1 and P2 are as shown in SEQ ID NO.4 and SEQ ID NO.5; the second probe is probe P3, and the sequence of the probe P3 is as shown in SEQ ID NO.

8.

2. The primer-probe composition according to claim 1, wherein The 5'-end part of the first probe is complementary to the sequence of the 5'-end of the downstream primer of the first primer pair; the 3'-end part of the first probe is complementary to the target sequence of the amplification product.

3. The primer-probe composition according to claim 1, characterized in that, The sequences of the third primer pair IC-F and IC-R are as shown in SEQ ID NO.9 and SEQ ID NO.10; the sequence of the third probe IC-P is as SEQ IDNO.

11.

4. The primer-probe composition according to claim 3, wherein The first probe, the second probe and the third probe are respectively labeled with different fluorescent markers; the fluorescent markers are selected from fluorescent dyes with maximum emission wavelengths of 518nm, 538-555nm, 574-575nm, 602-615nm, 640nm, 660-667nm or 690nm.

5. Use of a primer-probe composition in the preparation of a spinal muscular atrophy (SMA) gene mutation detection preparation and / or kit, characterized in that, The primer-probe composition is the primer-probe composition according to any one of claims 1-3.

6. A preparation and / or kit for detecting spinal muscular atrophy (SMA) gene mutations, characterized in that, It includes the primer-probe composition according to any one of claims 1-4.

7. A spinal muscular atrophy (SMA) gene mutation detection preparation and / or kit according to claim 6, characterized in that, It further includes a universal digital PCR reaction premix, an enhancer, a blank control, a negative control and a positive control.

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